Heart disease is the leading cause of mortality in United States and causes more deaths than all cancers combined. Coronary heart disease (or ischemic heart disease) is the most common type of heart disease and is inexplicably tied to pH regulation in the heart. However, the mechanisms of pH regulation in cardiomyocytes remain incompletely understood. The critical knowledge gaps stem from the fact that the proton neutralizing buffers such as HCO3- and Cl- homeostasis are not well understood. Recently, we have identified and cloned different isoforms of a solute carrier, Slc26a6, from cardiac myocytes. Slc26a6 is the predominant Cl-/HCO3- and Cl-/OH- exchanger in the heart. We demonstrated that Slc26a6 mediates electrogenic Cl-/HCO3- exchange activities in both atrial and ventricular myocytes. Our findings raise the possibility that Slc26a6 may represent the predominant Cl-/HCO3- regulatory mechanism in the heart. We have obtained exciting data to support the critical roles of Slc26a6 in cardiac excitability and contractility. We documented that null deletion of Slc26a6 in mice results in shortened action potentials (APs), fragmented QRS complexes on ECG and worsening cardiac function compared to wild type littermates. In addition, we have identified and characterized two isoforms of human SLC26A6 in human heart, which are also electrogenic, akin to mouse cardiac Slc26a6. Taken together, we hypothesize that the electrogenic Cl-/HCO3- exchange activities of Slc26a6 are critical not only for Cl- homeostasis, but also for the regulation of cardiac acid-base balance. Since cardiac excitability and contractility are intricately dependent on the pH, we further hypothesize that Slc26a6 plays critical roles in cardiac excitability and contractility, and the activity of Slc26a6 is cardioprotective against ischemia/reperfusion (I/R) injury. Thus, we predict that Slc26a6 may represent a novel Cl- and acid-base regulation mechanism in the heart that can be exploited to ameliorate cellular damages from cardiac ischemia. We will test our hypothesis using multidisciplinary approaches including functional electrophysiological recordings, imaging, biochemical, molecular and genetic approaches as well as in vivo functional studies. Wild type and cardiac- specific Slc26a6 knockout mouse model as well as human cardiomyocytes will be tested. Three specific aims are: 1. To test the roles of Slc26a6 in the regulation of intracellular pH (pHi) and Cl- (Cl-i) in cardiomyocytes. We will measure pHi and Cl-i of cardiomyocytes using membrane targeting pH and Cl- fluorescent biosensors. Patch-clamp recording coupled with pHi and Cl-i measurement will be performed to directly test the effects of the electrogenic Slca6a6 currents on pHi and Cl-i. 2. To determine the molecular and cellular mechanisms of Slc26a6 in the regulation of cardiac function. Since Slc26a6 is electrogenic, we will determine mechanisms of Slc26a6 in the regulation of AP profiles, Ca2+ transient and contractility of cardiomyocytes. 3. To determine the roles of Slc26a6 in cardiac function in vivo using normal and ischemia/reperfusion (I/R) mouse models. Our studies will unravel a missing molecular link between Cl- homeostasis and pHi regulation in the heart. The anticipated results will provide the first evidence demonstrating the roles of Slc26a6 in cardiac Cl- homeostasis, pHi regulation, and cardiac function under physiological and pathological conditions. At the translational level, Slc26a6 may represent a novel therapeutic target for cardioprotection in cardiac ischemia.